Oxalate Exudation by Taro in Response to Al1
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Plant Physiol. (1998) 118: 861–865
Oxalate Exudation by Taro in Response to Al1
Zhong Ma2 and Susan C. Miyasaka*
University of Hawaii-Manoa, Hawaii Branch Station, 461 West Lanikaula Street, Hilo, Hawaii 96720
grow at high Al concentrations in nutrient solutions (Mi-
Roots of taro (Colocasia esculenta [L.] Schott cvs Bun-long and yasaka et al., 1993a) that would be toxic to many other crop
Lehua maoli) exuded increasing concentrations of oxalate with species. Taro must have a mechanism to avoid Al toxicity,
increasing Al stress. This exudation was a specific response to excess because it does not accumulate high concentrations of Al in
Al and not to P deficiency. Addition of oxalate to Al-containing its shoots (Miyasaka et al., 1993b).
solutions ameliorated the toxic effect of Al. Taro contains substantial quantities of oxalate (Standal,
1983), as do the majority of higher plants (82 of 93 orders)
(Zindler-Frank, 1976). The ubiquity of oxalate in plants has
Al toxicity is a major factor limiting plant growth in raised questions about its roles. Proposed functions of ox-
many regions of the world (Kamprath, 1984), yet some alate include protection from insects and foraging animals
species or cultivars within species are able to grow in through toxicity and/or unpalatability, osmoregulation,
Al-toxic soils (Foy, 1988). A mechanism proposed to func- and regulation of Ca levels in plant organs and tissues
tion in Al tolerance by plants is the exudation of Al- (Libert and Franceschi, 1987).
chelating organic acids into the rhizosphere. Evidence that We propose that the secretion of oxalate is a mechanism
supports this hypothesis is as follows: (a) Al-stimulated by which taro avoids Al toxicity. We characterized the
exudation of malate occurs in Al-tolerant wheat cultivars exudation of oxalate from taro roots into the rhizosphere in
(Delhaize et al., 1993; Basu et al., 1994); (b) oxalate is response to excess Al.
secreted in response to Al by buckwheat, a known Al-
accumulating species; (c) citrate exudation is increased by
exposure to Al in an Al-tolerant snapbean cultivar (Mi- MATERIALS AND METHODS
yasaka et al., 1991) and in Al-tolerant corn genotypes (Pel-
Tissue-cultured plantlets of two taro (Colocasia esculenta
let et al., 1995); and (d) overexpression of a bacterial citrate
L.) cultivars, Bun-long and Lehua maoli, were obtained
synthase gene in transgenic tobacco increased cytoplasmic
from a commercial laboratory (Paradise Propagation, Hilo,
citrate production, increased the release of citrate extracel-
HI). Plants were ready for experiments when they were
lularly, and reduced the inhibition of root growth by Al (de
about 10 cm tall, with an average of six leaves and approx-
la Fuente et al., 1997).
imately 5-cm roots.
In addition, exogenous application of organic acids (e.g.
Plantlets were grown in nutrient solution under aseptic
malate, citrate, and oxalate) to nutrient solutions contain- conditions to quantitatively estimate their production of
ing toxic levels of Al has been shown to protect root growth organic acids. Microbial contamination was monitored by
and reduce Al toxicity in several crop species, such as plating samples onto nutrient agar, and only negligible
wheat (Ownby and Popham, 1989; Delhaize et al., 1993; contamination was detected.
Basu et al., 1994; Ryan et al., 1995; Pellet et al., 1996), The macronutrient concentrations in a modified Stein-
sorghum (Shuman et al., 1991), and corn (Pellet et al., 1995). berg solution were (in mm): NH4-N, 1.2; NO3-N, 3.6; P, 0.1;
According to Hue et al. (1986), malate is a moderate detox- K, 1.2; Ca, 1.0; Mg, 0.4; and S, 0.7. The micronutrients were
ifier, whereas both citrate and oxalate are strong detoxifiers (in mm): Mn, 2; B, 6; Zn, 1; Cu, 0.5; Mo, 0.1; and Fe as
of Al. Fe-N,N9-ethylenebis[2-(2-hydroxyphenyl)-glycine], 10 (Mi-
Taro (Colocasia esculenta L.), a tropical root-crop species, yasaka et al., 1993a). All nutrient solutions were autoclaved
is naturally tolerant of excess Al (Miyasaka et al., 1993a). except AlCl3, which was filter sterilized through 0.2-mm-
Two taro cultivars, Bun-long and Lehua maoli, are able to pore-diameter syringe filters to avoid Al precipitation
caused by pH changes during autoclaving.
1
This research was supported by the U.S. Department of Agri- The pH of the basal nutrient solution was adjusted to 4.0
culture under the Cooperative State Research, Education and Ex- with 0.1 m HCl and autoclaved. After the addition of
tension Service (special grant agreement no. 94-34135-0646), man- filter-sterilized, 0.09 m AlCl3 solution, pH 3.87, solutions
aged by the Pacific Basin Administrative Group. This is journal were again adjusted to pH 4.0 using 0.1 m NaOH. The final
series no. 4363 of the College of Tropical Agriculture and Human
solution pH was also recorded after harvest.
Resources, University of Hawaii-Manoa.
2
Present address: Department of Horticulture, The Pennsylva-
Before treatment plantlets were rinsed three times each
nia State University, 103 Tyson Building, University Park, PA with 15 mL of sterile deionized water to remove substances
16802– 4200. that may have accumulated on root surfaces during the
* Corresponding author; e-mail miyasaka@hawaii.edu; fax previous growth period. Plantlets of similar weights were
1– 808 –974 – 4110. grouped together. All experiments followed a randomized
861
Downloaded on December 17, 2020. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.862 Ma and Miyasaka Plant Physiol. Vol. 118, 1998
complete-block design to remove variability attributable to 900 mm Al and oxalic acid (0, 300, 600, 900, 1200, and 1500
plant size, light conditions, and temperature. Vessels were mm). Each plantlet was grown in 15 mL of solution in a test
kept in a growth chamber at 22°C to 28°C, 65% RH, 24 h of tube under aseptic conditions. Initial fresh weight of cv
light, and a light intensity of 45 mmol photons m22 s21. Bun-long was 0.40 6 0.07 g, and that of cv Lehua maoli was
0.98 6 0.16 g.
At the end of 14 d, when visible differences in root
Ion Chromatography
growth attributable to treatments were observed, plantlets
Samples of the solution culture medium were analyzed were harvested and root length was determined. Relative
for oxalate and other organic acids using HPLC (DX 500 root length was calculated as the fraction of root length in
chromatography system, Dionex, Sunnyvale, CA) with an various treatment solutions divided by that in controls
anion self-regenerating suppressor (4 mm, model ASRS-I, (plants grown in basal nutrient solution only).
Dionex). For oxalate analysis, an anion-exchange analytical
column and a guard column (both 4 mm, models IonPac Role of Low P in Oxalate Exudation
AS4A-SC and AG4A-SC, Dionex) were used with an eluent
of 22 mm sodium borate and boric acid at a flow rate of 2.0 Al can induce P deficiency in plants through its com-
mL min21. The columns were also used to reconfirm iden- plexation of phosphate ions, and plants are known to re-
tification of oxalate and for analyses of other organic acids. spond to P deficiency by exudation of organic acids (Lipton
For analyses of oxalate, succinate, and malate, 50 mm et al., 1987). To investigate whether oxalate exudation was
NaOH was the eluent, and for citrate, 100 mm NaOH was related to low P or to Al stress, tissue-cultured plantlets
the eluent, both at a flow rate of 1.0 mL min21. Concentra- were grown under sterile conditions in the presence or
tions of organic acids were determined via measurement of absence of 900 mm Al and three levels of P (0, 0.3, and 100
electrical conductivity using a conductivity detector (mod- mm). Treatments were a complete factorial combination of
el CD20, Dionex). two cultivars, two Al levels, and three P levels. Each treat-
ment was replicated five times, with one plantlet grown in
a culture tube containing 15 mL of nutrient solution per
Time Course of Al-Induced Oxalate Exudation replicate. The initial weight of cv Bun-long averaged 1.6 6
Tissue-cultured plantlets of the taro cvs Bun-long and 0.2 g, and that of cv Lehua maoli averaged 2.2 6 0.61 g. One
Lehua maoli were grown in sterile culture for 10 d in the week after exposure to treatments, solution samples were
basal nutrient solution with or without 900 mm Al. Four collected and analyzed for oxalate.
replicates were used in a factorial combination of two Al
levels and two taro cultivars. For each treatment two plant- Statistical Analyses
lets were grown in 50 mL of treatment solution. Initial fresh Statistical analyses of the data were conducted using SAS
weight of cv Bun-long averaged 6.79 6 0.75 g, and that of (Statistical Analysis Systems Institute, 1982). In the time-
cv Lehua maoli was 4.54 6 0.85 g. course trial, analysis of variance was used for a repeated-
Samples from the growth medium were taken on d 3, 5, measures design. In the other experiments, two-way or
7, and 10 after treatments were imposed. At each sampling three-way analysis of variance was used. In the dose-
time, 1 mL of solution was withdrawn from each growth response experiment, single degree-of-freedom contrasts
vessel, diluted with 1 mL of sterile deionized water, and were used to evaluate linear, quadratic, and cubic effects of
then stored in a freezer until analyses for oxalate. Al on oxalate exudation. A P value of 0.05 or less was
considered statistically significant.
Dose Response of Al-Induced Oxalate Exudation
RESULTS
Two taro cultivars were exposed to four initial Al levels
(0, 300, 600, and 900 mm) in the basal nutrient solution. Time Course of Al-Induced Oxalate Exudation
Treatments were a complete factorial of two taro cultivars In the control treatments, both cultivars released only
and four Al levels, with five replicates. For each treatment small concentrations of oxalate, and the exudation changed
three plantlets were grown in a vessel containing 100 mL of only slightly over time (Fig. 1). The addition of 900 mm Al
treatment solution. Initial fresh weight of cv Bun-long av- significantly stimulated oxalate excretion from roots of
eraged 11.9 6 0.61 g, and that of cv Lehua maoli averaged both taro cultivars (P 5 0.0001), with a linear increase in the
7.3 6 0.60 g. Solution samples were taken for the analyses cumulative amount up to 7 d after initiation of treatment
of oxalate and other organic acids (succinate, malate, and (Fig. 1). No significant difference in oxalate exudation was
citrate) 1 week after exposure to the Al treatments. found between the cultivars (P 5 0.12).
Final solution pH was in the range of 3.5 to 4.2, with no
Amelioration of Al Toxicity by Oxalate significant differences in final solution pH attributable to
cultivars or Al levels (P 5 0.56 and 0.47, respectively).
Roots were removed and plantlets were grown in sterile
nutrient solution for 2 d to allow healing of wounds. Plant-
Dose Response of Al-Induced Oxalate Exudation
lets were then transferred to seven treatment solutions,
with five replicates per treatment. Treatments included Increasing Al concentrations from 0 to 900 mm signifi-
basal nutrient solutions and basal nutrient solutions plus cantly increased exudation of oxalate into the rhizosphere
Downloaded on December 17, 2020. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.Oxalate Exudation by Taro in Response to Al 863
Figure 1. Exudation of oxalate from roots (on a fresh-weight basis) of
taro cvs Bun-long (BL) and Lehua maoli (LM) at Al levels of 0 (Al0) Figure 3. Effect of exogenously applied oxalate on relative root
and 900 (Al900) mM over a period of 10 d. lengths of taro cvs Bun-long (BL) and Lehua maoli (LM) grown at 900
mM Al over a period of 14 d.
from both cultivars (P 5 0.0001; Fig. 2). No significant
cultivar difference was detected in oxalate exudation in grown in the presence of 900 mm Al (linear effect: P 5 0.03;
response to increasing Al levels (P 5 0.65; Fig. 2). Accu- Fig. 3). Oxalate concentrations greater than 900 mm began
mulation of oxalate in the growth medium was linear for cv to reverse the ameliorative effect on Al-induced inhibition
Bun-long over the range of solution Al levels tested (P 5 of root growth (quadratic effect: P 5 0.03; Fig. 3). Taro cv
0.0002; Fig. 2), whereas for cv Lehua maoli, the increase of Lehua maoli had significantly longer relative root lengths
oxalate concentration with increased Al levels followed a compared with cv Bun-long (P 5 0.01; Fig. 3). For cv Lehua
cubic trend (P 5 0.0043; Fig. 2). maoli, an oxalate concentration of 600 mm was sufficient to
No detectable amounts of citrate, malate, or succinate restore relative root length to that of the controls, whereas
were found in the nutrient solution. The final solution pH for cv Bun-long, 900 mm oxalate was required to restore
ranged from 3.8 to 4.2 for cv Bun-long and from 3.6 to 3.8 relative root length to 88% of the controls.
for cv Lehua maoli, with a significant difference attribut- The final solution pH for cv Bun-long varied from 3.9 to
able to cultivar (P 5 0.0001), but none attributable to Al 4.2, and that of cv Lehua maoli ranged from 3.6 to 4.1, with
treatment (P 5 0.08). a significant difference attributable to cultivar (P 5 0.004).
Amelioration of Al Toxicity by Oxalate Role of Low P in Oxalate Exudation
Exogenously added oxalate up to 900 mm significantly There were no significant differences in oxalate exuda-
increased relative root lengths for both taro cultivars tion between P treatments for either cultivar, whereas the
presence of Al in solution significantly increased exudation
of oxalate (Table I). Much higher concentrations of oxalate
were exuded by cv Bun-long in response to 900 mm Al
relative to cv Lehua maoli, resulting in a significant inter-
action between cultivar and Al treatments (Table II).
Final solution pH ranged from 3.9 to 5.7 for cv Bun-long
and from 3.7 to 4.5 for cv Lehua maoli, with significant
Table I. Effect of P and Al levels on the exudation of oxalate from
roots of two taro cultivars over 7 d
Values are means (6SE).
Oxalate Exudation
cv
0 mM P 0.3 mM P 100 mM P
mg g21 root fresh wt
Bun-long
0 mM Al 48.2 (9.9) 64.2 (12.7) 59.6 (14.0)
900 mM Al 535.9 (111.3) 693.7 (148.8) 523.6 (96.6)
Lehua maoli
Figure 2. Effect of increasing total Al levels in nutrient solution on
0 mM Al 32.1 (16.4) 19.8 (8.7) 30.7 (6.7)
the exudation of oxalate from roots (on a fresh-weight basis) of taro
900 mM Al 188.2 (25.1) 284.2 (97.8) 284.2 (123.7)
cvs Bun-long (BL) and Lehua maoli (LM) over a period of 7 d.
Downloaded on December 17, 2020. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.864 Ma and Miyasaka Plant Physiol. Vol. 118, 1998
Table II. Analysis of variance probabilities for P, Al, and cultivar the growth of taro plantlets in a sterile, nutrient medium as
effects on exudation of oxalate a means to assay for cultivar differences.
P 0.4200
Taro cv Bun-long exuded significantly greater concentra-
Al 0.0001 tions of oxalate relative to cv Lehua maoli at 900 mm Al
Cultivar 0.0001 (Table I). These results do not agree with previous research
P 3 Al 0.4300 in which Al-tolerant genotypes of corn (Pellet et al., 1995)
P 3 cultivar 0.6400 and wheat (Delhaize et al., 1993; Basu et al., 1994) exuded
Al 3 cultivar 0.0005 more Al-chelating organic acids into the rhizosphere com-
P 3 Al 3 cultivar 0.7100 pared with Al-sensitive genotypes in the presence of excess
Replicate 0.0150 Al. Our results indicate that a second Al-tolerance mecha-
nism must operate in cv Lehua maoli exposed to very high
Al levels.
differences attributable to cultivar (P 5 0.0001). P treat- In previous research, water-soluble oxalate concentra-
ments did not significantly affect solution pH (P 5 0.91), tions decreased within roots of taro exposed to Al (Calisay,
but treatments with added Al had significantly lower so- 1996). In our experiment using 900 mm Al, cv Bun-long
lution pH (P 5 0.0001). Final solution pH levels greater exuded oxalate at 5.1 g kg21 root dry weight and cv Lehua
than 4.3 were found only in treatment solutions not con- maoli exuded oxalate at 3.1 g kg21 root dry weight. In an
taining Al. experiment using 890 mm Al (Calisay, 1996), the water-
soluble oxalate concentration in roots decreased 2.0 and
2.2 g kg21 root dry weight for cvs Bun-long and Lehua
DISCUSSION
maoli, respectively. The greater concentrations of oxalate
Al-stimulated oxalate exudation from roots of two taro exuded from taro roots compared with the decrease in
cultivars was found within 3 d after the initiation of Al water-soluble oxalate levels within taro roots might have
treatments (Fig. 1). The increase in cumulative oxalate con- been caused by differences in the size of the root systems or
centration was linear up to 7 d after exposure to Al. Our to de novo synthesis of oxalate.
long-term results confirm the findings of Ma et al. (1997), in To our knowledge, this report is the first to demonstrate
which short-term exposure of buckwheat roots to AlCl3 that a non-Al-accumulating crop species exudes oxalate
induced oxalic acid excretion in 30 min and a linear in- into the rhizosphere in response to excess Al. Our findings
crease in the amount excreted over 12 h. demonstrate that there is no linkage between exudation of
Increasing Al levels in solution significantly increased oxalate and internal accumulation of an Al-oxalate com-
oxalate concentrations released from roots of two taro cul- plex. Because high levels of Al in the edible portions of a
tivars (Fig. 2). Again, our results agree with those of Ma et plant may be detrimental to the health of consuming hu-
al. (1997), who showed that Al-induced exudation of ox- mans or animals, it is important to establish that exudation
alate by buckwheat increased with increasing external Al of oxalate by roots does not result in increased accumula-
levels. tion of Al in shoots.
Addition of oxalate to solutions containing 900 mm Al Our data are consistent with the hypothesis that oxalate
ameliorated the Al-induced inhibition of root elongation in is excreted by taro into the rhizosphere, where it detoxifies
both taro cultivars (Fig. 3). Similar amelioration of Al tox- Al, and that an additional mechanism of Al tolerance is
icity by exogenously applied oxalate was found for sor- found at very high Al levels. Our tissue-culture assay
ghum (Shuman et al., 1991) and cotton (Hue et al., 1986). provides an ideal method to further characterize these
The reversal of the ameliorative effect of oxalate at concen- Al-tolerance mechanisms, as shown by the correlation be-
trations greater than 900 mm (Fig. 3) could be caused by a tween Al tolerance of taro cultivars in soil and the tissue-
toxic effect of oxalate alone on root elongation. A detrimen- culture medium.
tal effect of oxalate was found in sorghum, in which 10 mm
ACKNOWLEDGMENTS
oxalate decreased the root length of seedlings by 80% in the
absence of Al (Shuman et al., 1991). The authors thank Dr. Michael Tanabe (University of Hawaii-
Taro cultivars exuded oxalate specifically in response to Hilo) for helpful suggestions and sharing of equipment, Joanne
excess Al and not to deficient levels of P. P levels in Lichty for generous sharing of laboratory facilities, and Drs. Wil-
liam Sakai (University of Hawaii-Hilo) and Chung-shih Tang for
solution had no significant effect on oxalate exudation
useful comments and insights concerning these experiments.
during a 1-week growth period (Table I). Our results con-
firm the findings of Ma et al. (1997), in which P deficiency
Received July 29, 1998; accepted August 5, 1998.
did not induce oxalate excretion by buckwheat.
Copyright Clearance Center: 0032–0889/98/118/0861/05.
In previous studies using both soil and nutrient solution,
cv Lehua maoli was found to be Al tolerant compared with
LITERATURE CITED
cv Bun-long at Al levels $ 890 mm (Miyasaka et al., 1993a;
Calisay, 1996). These cultivar differences were confirmed Basu U, Godbold D, Taylor GJ (1994) Aluminum resistance in
in our experiment with exogenous application of oxalate, in Triticum aestivum associated with enhanced exudation of malate.
J Plant Physiol 144: 747–753
which cv Lehua maoli was found to have significantly Calisay MG (1996) Differential response of taro (Colocasia esculenta
longer relative root lengths compared with cv Bun-long in L.) cultivars to aluminum toxicity. PhD thesis. University of
the presence of 900 mm Al (Fig. 3). These results validate Hawaii, Honolulu
Downloaded on December 17, 2020. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.Oxalate Exudation by Taro in Response to Al 865
de la Fuente JM, Ramirez-Rodriguez V, Cabrera-Ponce JL, Miyasaka SC, Webster CM, Okazaki EN (1993b) Differential re-
Herrera-Estrella L (1997) Aluminum tolerance in transgenic sponse of two taro cultivars to aluminum. II. Plant mineral
plants by alteration of citrate synthesis. Science 276: 1566–1568 concentrations. Commun Soil Sci Plant Anal 24: 1213–1229
Delhaize E, Ryan PR, Randall PJ (1993) Aluminum tolerance in Ownby JD, Popham HR (1989) Citrate reverses the inhibition of
wheat (Triticum aestivum L.). II. Aluminum-stimulated excretion wheat root growth caused by aluminum. J Plant Physiol 135:
of malic acid from root apices. Plant Physiol 103: 695–702 588–591
Foy CD (1988) Plant adaptation to acid, aluminum-toxic soils. Pellet DM, Grunes DL, Kochian LV (1995) Organic acid exuda-
Commun Soil Sci Plant Anal 19: 959–987 tion as an aluminum-tolerance mechanism in maize (Zea mays
Hue NV, Craddock GR, Adams F (1986) Effect of organic acids on L.). Planta 196: 788–795
aluminum toxicity in subsoils. Soil Sci Soc Am J 50: 28–34 Pellet DM, Papernik LA, Kochian LV (1996) Multiple aluminum-
Kamprath EJ (1984) Crop responses to lime on soils in the tropics. resistance mechanisms in wheat: roles of root apical phosphate
In F Adams, ed, Soil Acidity and Liming, Ed 2. Agronomy and malate exudation. Plant Physiol 112: 591–597
Monograph No. 12. American Society of Agronomy, Madison, Ryan PR, Delhaize E, Randall PJ (1995) Malate efflux from root
WI, pp 349–368 apices and tolerance to aluminum are highly correlated in
Libert B, Franceschi VR (1987) Oxalate in crop plants. J Agric wheat. Aust J Plant Physiol 22: 531–536
Food Chem 35: 926–938 Shuman LM, Wilson DO, Ramseur EL (1991) Amelioration of
Lipton DS, Blanchar RW, Blevins DG (1987) Citrate, malate, and aluminum toxicity to sorghum seedlings by chelating agents.
succinate concentration in exudates from P-sufficient and J Plant Nutr 14: 119–128
P-stressed Medicago sativa L. seedlings. Plant Physiol 85: 315–317 Standal BR (1983) Nutritive value. In JK Wang, ed, Taro: A Review
Ma JF, Zheng SJ, Matsumoto H (1997) Detoxifying aluminum of Colocasia esculenta and Its Potentials. University of Hawaii
with buckwheat. Nature 390: 569–570 Press, Honolulu, pp 141–147
Miyasaka SC, Buta JG, Howell RK, Foy CD (1991) Mechanism of Statistical Analysis Systems Institute (1982) SAS User’s Guide:
aluminum tolerance in snapbeans. Plant Physiol 96: 737–743 Statistics. SAS Institute, Cary, NC
Miyasaka SC, Webster CM, Hue NV (1993a) Differential response Zindler-Frank E (1976) Oxalate biosynthesis in relation to photo-
of two taro cultivars to aluminum. I. Plant growth. Commun Soil synthetic pathway and plant productivity: a survey. Z Pflanzen-
Sci Plant Anal 24: 1197–1211 physiol 80: 1–13
Downloaded on December 17, 2020. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.You can also read
